Peptide-based carrier devices for stellate cells

ABSTRACT

The present invention relates to a compound comprising a carrier molecule, said carrier molecule being linked to a further molecule, said further molecule being at least one cyclic peptide, said cyclic peptide comprising in the cyclic peptide portion thereof at least one sequence encoding a cell receptor recognising peptide (RRP) and with the proviso that the compound is not a naturally occuring receptor agonist or antagonist. Preferably, the RRP is of a receptor specific for Hepatic Stellate Cells (HSC) or a receptor that is upregulated on HSC during disease. In particular, the RRP may be of a receptor selected from the group of PDGF receptor, collagen type VI receptor, cytokine receptor(s) such as TGFβ, IFNα and interleukin 1β. Preferably, the cyclic portion of the cyclic peptide comprises at least the amino acid sequence RGD or KPT. The compounds can be used as an active targeting ingredient for manufacturing a pharmaceutical composition for therapy, prophylaxis or diagnosis of a disease selected from the group consisting of fibrotic disease, sclerotic disease and chronic or acute inflammatory processes such as glomerulosclerosis, interstitial fibrosis, lung fibrosis, atherosclerosis, rheumatoid arthritis, Crohns disease, colitis ulcerosa, glomerulonephritis and sepsis, in particular for targeting HSC. The invention also relates to pharmaceutial compositions comprising the above compound(s).

This application is the National Phase of International ApplicationPCT/NL98/00579 filed Oct. 8, 1998 which designated the U.S. and thatInternational Application was published under PCT Article 21(2) inEnglish.

BACKGROUND OF THE INVENTION

The hallmark of fibrosis is the excessive deposit of extracellularmatrix components caused by an increased synthesis and decreaseddegradation of matrix proteins, predominantly collagen type I and III.This process of fibrosis can occur in all kinds of organs such as thekidney (glomerulosclerosis or interstitial fibrosis), the skin (scarformation), the lung and also in the liver, where the end-stage of liverfibrosis is referred to as cirrhosis. The process also shares manycharacteristics with the formation of atherosclerotic plaques inarteries. Liver fibrosis leads to a deterioration of liver function, andeventually in complete liver failure, which is lethal if untreated. Theprocess can be elicited by viruses (Hepatitis A, B and C), alcoholconsumption, genetic disorders, or by chronic exposure to hepatotoxicagents. The incidence of this disease is very variable depending on thecountry. In the period 1985-1989, the incidence of liver cirrhosis inThe Netherlands was 3.90 per 100,000 habitants per year, whereas thisincidence in, for instance, France and Germany was 11.9 respectively12.4. To date, no effective pharmacotherapeutic intervention isavailable for this disease. In the past decades liver transplantationhas become a serious option for many patients but the costs, theavailability of donor livers and the traumatic event of thetransplantation itself hamper the application of such an operation ingeneral practice. Pharmacological intervention would be a better option.

Hepatic stellate cells (HSC), also called Ito cells or fat storing cellsstrongly proliferate during the progression of the disease and theysubsequently transform into myofibroblasts (MF). These cells are themajor producers of collagens, glycoproteins, and proteoglycans in adiseased liver. Moreover, HSC and MF produce an array of mediators whichactivate other hepatic and inflammatory cells thus enhancing thefibrotic process. Therefore, HSC are an important target foranti-fibrotic therapy. However, in vivo studies indicate thatanti-fibrotic drugs are not efficiently taken up by HSC and as aconsequence, most drugs which showed potent anti-fibrotic activity invitro, failed to exert any effect in vivo. At high doses such drugsoften induce many side effects caused by extrahepatic distribution ofthe drug. Cell specific delivery is an option to solve these problems.This can be accomplished by coupling drugs to carrier molecules, whichare selectively taken up by the target cells. Liposomes are well knowndrug carriers but modified proteins can also be applied. Cell specificdelivery of therapeutic and diagnostic agents to hepatocytes,endothelial and Kupffer cells has already been achieved by modificationof the sugar moieties of proteins or polymers. Coupling of galactose to,for instance, human serum albumin (HSA) leads to a specific accumulationof this neoglycoprotein in hepatocytes whereas addition of mannose toalbumin causes uptake into Kupffer or endothelial cells. Increasing thenet negative charge (for instance by succinylation of amine groups)results in uptake of the protein into endothelial cells via scavengerreceptors. For a comprehensive review on carrier devices for cellspecific delivery of drugs see D. K. F. Meijer and G. Molema, Sem. inLiver Dis. 15: 202-256, 1995. The benefits of such carrier devices forthe development of novel pharmacotherapeutic interventions for variousdiseases has been well recognized. However, a specific carrier for drugsto HSC, the most important cell in the pathogenesis of liver fibrosis,has not been found yet.

SUMMARY OF THE INVENTION

The invention describes novel drug carriers which specificallyaccumulate in hepatic stellate cells (HSC). These carriers can be usedfor the targeting of all kinds of therapeutic agents, preferablyanti-fibrotic drugs to HSC. The carriers may also be applied for thevisualization of HSC for diagnostic purposes. The basis of the inventionlies in the coupling of small cyclic proteins (oligopeptides), thatcontain specific receptor recognising peptides (RRPs) to soluble orparticle type carriers (core carriers). The use of such a conjugate as atool for targeting purposes has not been described. The target-receptorsfor these neo proteins, neo oligopeptides or oligopeptide carrierconstructs are specific for HSC or are upregulated upon this cell typeduring the course of the disease. In the present study human serumalbumin (HSA) is applied as the core-carrier, but the invention is notrestricted to a specific protein or polymer. Each molecule withattachment sites for peptides is applicable as a carrier to HSC. Theinvention describes conjugates which bind to the platelet derived growthfactor (PDGF)-receptor and corijugates which attach to the collagen typeVI receptor. Both types of receptors are present in relatively highamounts upon HSC and are well characterized. The respectivereceptor-binding ligands are known.

Since these receptors are also upregulated in renal mesangial cells aswell as fibroblasts in various organs during glomerulosclerosis,interstitial fibrosis, lung fibrosis or atherosclerosis, and since thesepathological processes are accessible for macromolecules it is assumedthat these carriers will also show a relative accumulation in these celltypes during the course of these diseases. The conjugates described heremay therefore also be applied as drug-carriers or carriers fordiagnostic markers and/or for treatment of the above mentioned diseases.

The proliferation of HSC during the process of fibrosis is an importantpathogenic factor. Cell-matrix interactions and the production of growthfactors such as PDGF play a pivotal role in this proliferative responseof HSC. Peptides which bind to the PDGF receptors or collagen type VIreceptors will block the binding of endogenous PDGF or will interferewith cell-matrix interactions and therefore the oligopeptides describedhere may also exert an antiproliferative activity and consequently theseoligopeptides may serve as anti-fibrotic or anti-sclerotic agentsthemselves.

Also other receptors may be targeted using this new approach.Transforming Growth Factor β(TGFβ), interleukin 1β (II 1β), and TumorNecrosis Factor α (TNFα) are other important mediators during chronicinflammatory processes and the receptors for these cytokines areupregulated upon HSC as well as upon endothelial cells and Kupffer cellsin the liver. The ligands for these receptors are well characterized andsimilar to the PDGF-receptor or collagen VI-receptor recognizingproteins, cyclic peptides recognizing the binding site for thesereceptors can be prepared and coupled to a core-protein such as albumin.A relative accumulation of these conjugates can be expected into thetarget cell expressing the particular receptors. Most of the cytokinescontain a RGD sequence (arg-gly-asp). This is the (putative) cellattachment site and in combination with additional amino acids it willdetermine the specificity for the individual cytokines and growthhormone receptors. Coupling this RGD sequence and accompanying aminoacids to a carriermolecule using the approach described here isfeasible. The invention also includes the preparation of oligopeptidesin which more than one receptor recognizing domain for the same receptorare combined and peptide constructs in which different receptorrecognizing domains for different types of receptors are combined. Theparticular oligopeptide constructs containing a single or more than onereceptor recognizing domain can be used as such, as intrinsic activesubstances but also for the preparation of drug conjugates (pro-drugs)and be employed to prepare larger drug carriers by coupling of theoligopeptides to either proteins, soluble and particulate polymericcarriers and lipoid carriers (liposomes, neolipoproteins, micelles) thatsubsequently can be used for covalent binding and/or inclusion orassociation of therapeutic agents for the purpose of cell-specific drugtargetting.

The application of such carriers is not limited to the treatment ordiagnosis of fibrotic processes but also to other chronic and acuteinflammatory processes such as, for instance, rheumatoid arthritis,Crohn's disease, colitis ulcerosa, glomerulonephritis and sepsis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1- a shows organ distribution of human serum albumin (HSA)conjugated with 10 to 12 cyclic oligopeptides recognizing thePDGF-receptor in normal rats.

FIG. 1- b shows organ distribution of human serum albumin (HSA)conjugated with 10 to 12 cyclic oligopeptides recognizing thePDGF-receptor in rats with liver fibrosis induced by bile duct ligation(3 weeks after the operation).

FIG. 1- c represents the organ distribution of unmodified HSA.

FIG. 2- a shows organ distribution of human serum albumin (HSA)conjugated with 10 to 12 cyclic oligopeptides recognizing the collagentype VI-receptor in normal rats.

FIG. 2- b shows organ distribution of human serum albumin (HSA)conjugated with 10 to 12 cyclic oligopeptides recognizing the collagentype VI-receptor in rats with liver fibrosis induced by bile ductligation (3 weeks after the operation).

FIG. 3- a shows that after intravenous administration of modifiedprotein, the albumin derivatives can be immunhistochemically detected ina non-parenchymal cell type of the liver using a polyclonal antibodyagainst albumin.

FIG. 3- b shows, as seen from the arrowheads, the modified albuminco-localizes with the marker for HSC (desmin).

FIG. 4 is a graph representing in vitro displacement of radiolabeledPDGF-BB from its receptor upon 3T3-fibroblasts by HSA-PDGFreceptor-binding peptide conjugates (pPB-HSA, closed blocks), HSA (openblocks) or uncoupled PDGF-receptor binding peptides (pPB, open circles).

FIG. 5 is a graph of the organ distribution of radiolabeled M6P^(x)-HSAin fibrotic rats (three weeks after bile duct ligation), 10 minutesafter intravenous administration of the modified HSA.

FIG. 6 is a graph of the binding and uptake of radiolabeled M6P₂₈—HSA inhuman liver tissue at the reported temperatures.

DETAILED DESCRIPTION OF THE INVENTION

A compound according to the invention comprises a carrier molecule, saidcarrier molecule being linked to a further molecule, said furthermolecule being at least one cyclic peptide, said cyclic peptidecomprising in the cyclic peptide portion thereof at least one sequencecontaining at least one specific receptor recognising peptide (RRP) andwith the proviso the compound is not a naturally occurring receptoragonist or antagonist. Suitably in such a compound according to theinvention the RRP is of a receptor specific for Hepatic Stellate Cells(HSC) or a receptor that is upregulated on HSC during disease.

The RRP can by way of example be the agonist or antagonist of a receptorselected from the group of receptors consisting of PDGF receptor,collagen type VI receptor, cytokine receptor such as TGFβ, TNFα andinterleukin 1β.

Suitably, when the RRP is of a collagen type VI receptor, cytokinereceptor such as TGFβ, TNFα and interleukin 1β, the cyclic portion ofthe cyclic peptide comprises at least the amino acid sequence RGD or KPT(lys-pro-thr) in the cyclic portion thereof. By way of example, thecyclic portion of the cyclic peptide comprises at least an amino acidsequence selected from X*YRGDYX* (Xaa(Xaa),-arg-gly-asp-(Xaa)_(n)-Xaa)and X*YKPJYX* (Xaa-(Xaa)-lys-pro-thr-(Xaa),-Xaa) wherein X* representsthe location of cyclisation and Y represents at least one amnio acid ora sequence of amino acids up to a length such that the receptor bindingcapacity of the cyclic peptide is retained. In an preferred embodiment,X* represents the location of attachment to the carrier molecule. In anembodiment illustrating the above, when the receptor agonist is of acollagen type VI receptor has a cyclic portion of the cyclic peptidecomprising the amino acid sequence X*GRGDSPX*(Xaa-gly-arg-gly-asp-ser-pro-Xaa). Suitably, it will comprise thesequence -cysteine-glycine-arginine-glycine-asparticaicd-serine-proline-cysteine. SEQ ID NO:1.

Suitably when the receptor agonist is of an interleukin 1 beta receptor,the cyclic peptide can comprise the amino acid sequence X*DKPTLX*(Xaa-asp-lys-pro-thr-lys-Xaa). SEQ ID NO:2.

Alternatively, when the receptor agonist is of PDGF receptor, the cyclicportion of the cyclid peptide can comprise the amino acid sequenceX*SRNLIDCX* (Xaa-ser-arg-asn-leu-ile-asp-cys-Xaa), wherein X* representsthe location of cyclisation. SEQ ID NO:3. In a preferred emodiment X*represents the location of attachment to the carrier molecule. Such acompound will bind to the PDGF receptor alha and beta subtypes.Suitably, it will comprise thesequence-cysteine-serine-arginine-asparagine-leucine-isoleucine-asparticacid-cysteine.

Only some crucial amino acids for the cyclic peptides are provided here.The oligopeptide may be elongated without causing a change in thecellular distribution pattern in vivo. Cyclisation of these peptides canbe achieved for example by a disulfide bond between both cysteinegroups. The free amine (α-amine) in one cysteine residue can be used tocouple the oligopeptide to the carrier molecule. For example to theamine groups in a core-molecule like albumin, using succinimide-acetylthioacetate (SATA). Coupling of more than one oligopeptide to albumincan be readily done. Attachment of the cyclic peptides to a carriermolecule via a biodegradable spacer, causing local release of the cyclicpeptides, is also feasible. The examples provided here describeconjugates with multiple oligopeptides per HSA molecule, leaving enoughfree reactive groups within the core-protein (hydroxyl, amine orsulphate groups) to attach additional drugs or other chemicals. Theseconjugates selectively accumulate in HSC of normal and diseased livers.

The cyclic portion of the cyclic peptide can suitably comprise multipleRRP sequences. The cyclic portion of the cyclic peptide can comprisemultiple RRP sequences directed at at least two different types ofreceptors. Obviously they can also be directed at the same type ofreceptor. Combinations of various receptor agonist sequences arenaturally also possible. Thus a compound according to the invention inany of the embodiments defined may comprise multiple cyclic peptidesdirected at the same or different subtypes of receptors or may comprisemultiple but similar oligopeptides that contain more than one identicalor different RRP sequence directed at the same receptor or differentreceptors on the particular cell type respectively. By way of example acompound according to the invention, wherein the carrier molecule islinked to more than one cyclic peptide can suitably comprise 5-15 cyclicpeptides as defined in any of the embodiments above.

A person skilled in the art will realise numerous types of carriermolecules can be applied. The carrier molecule can suitably be selectedfrom endogenous plasma proteins e.g. albumin, lactoferrin, alkalinephosphatase, superoxide dismutase, alpha2 macroglobulin and fibronectin.They are to be pharmaceutically acceptable and of a size such that theypreferably are not lost due to the renal excretion thereof. Suchcompounds are suitably larger than 5000 Daltons. Suitable examples ofthe carrier molecule can be selected from the group of carrier moleculesconsisting of proteins, oligo or polypeptides, immunoglobulins or partsthereof, oligonucleotides, disaccharides, polysaccharides, biodegradablesynthetic polymers, liposomes, lipid particles, biocompatible polymersin the form of microspheres or nanoparticles. Quite suitably in acompound according to this aspect of the invention the carrier moleculeis the endogenous plasma protein albumin. The immunoglobulins can bemono or polyclonal. Parts of immunoglobulins can comprise Fab′ fragmentsor single chain Ig. Humanised antibodies and bispecific antibodies areenvisaged. In the case of human administration carriers that occurnaturally in humans are preferred. For the sake of easy linkage of thecarrier molecule to the cyclic peptide the carrier molecule preferablycomprises free reactive groups such as hydroxyl, amine or sulphate. Thecarrier molecule can suitably be linked to the cyclic peptide via abiodegradable spacer. The carrier molecule can itself be a drug, theactivity of which is not impaired by linking the cyclic peptide to it.

In an alternative embodiment of the invention the carrier molecule inthe compound can comprise additional drugs or chemicals linked thereto.

The invention also covers a pharmaceutical composition comprising acompound according to any of the aforementioned embodiments as targetingingredient and any pharmaceutically acceptable carrier. A pharmaceuticalcomposition according to the invention comprises a compound in any ofthe embodiments mentioned above as pharmaceutically active ingredient incombination with any pharmaceutically acceptable additional carrier. Inan alternative embodiment the pharmaceutical composition can furthercomprise a drug biodegradably attached to the compound. It is alsopossible for the compound to further comprise a diagnostic markerattached thereto. A pharmaceutical composition according the inventionwill be in a pharmaceutical dosage form. Such a dosage form can comprisesprayable, injectable or infusable solutions or solids or dosage formsfor pulmonary or other administration routes. Also a pharmaceuticalcomposition according to the invention can be in a topical form. In thecase of parenteral administration a systemically acceptable form shouldbe composed. This means it can enter the bloodstream without causingclotting or inadmissibly toxic reactions.

The invention is also directed at application of a compound according tothe invention in any of the abovementioned embodiments as activetargeting ingredient for manufacturing a pharmaceutical compositionaccording to the invention for therapy, prophylaxis or diagnosis ofchronic diseases. Examples from this group consist of fibrotic disease,sclerotic disease and chronic or acute inflammatory processes such asglomerulosclerosis, interstitial fibrosis, atherosclerosis, rheumatoidarthritis, Crohns disease, colitis ulcerosa, glomerulonephritis, lungfibrosis and sepsis. Suitably use of a compound according to theinvention as active targeting ingredient for manufacturing apharmaceutical composition according to the invention for therapy,prophylaxis or diagnosis of a disease related to proliferation of HSC isalso envisaged as forming a particularly useful application to becovered by the invention. A method of targeting HSC, said methodcomprising administration in a pharmaceutically acceptable amount andform of a compound or a pharmaceutical composition according to theinvention to a subject or a tissue sample of a subject is covered by theinvention. The person skilled in the art will adjust the dosage to beapplied to the manner of application, see, weight, state of health etcof the subject to which administration is to occur. Administration canocur in any manner known per se for administration of medicament.

The invention also covers a method of therapy, diagnosis or prophylaxisof a disease related to HSC, said method comprising administration in apharmaceutically acceptable amount and form of a compound or apharmaceutical composition according to the invention to a subject or atissue sample of a subject. Particularly such disease can be oneselected from the group consisting of fibrotic disease, scleroticdisease and chronic or acute inflammatory processes such asglomerulosclerosis, interstitial fibrosis, lung fibrosis,atherosclerosis, rheumatoid arthritis, Crohns disease, colitis ulcerosa,glomerulonephritis and sepsis. The method comprises administration in apharmaceutically acceptable amount and form of the compound orpharmaceutical composition according to the invention to a subject or atissue sample of a subject The person skilled in the art will adjust thedosage to be applied to the manner of application, size, weight, stateof health etc of the subject to which administration is to occur.Administration can occur in any manner known per se for administrationof therapeutic agents.

This further aspect of the invention will be illustrated but not limitedin the following examples.

EXAMPLE 1

Normal rats and rats with liver fibrosis (3 weeks after bile ductligation) received an intravenous injection of 10 mg/kg b.w. PDGFreceptor-binding peptides conjugated with HSA. Based upon the results ofthe organ distribution studies with radiolabeled conjugates (FIG. 1),rats were sacrificed after ten minutes and samples of the liver and bone(from ribs, front paw, rear paw and the back) were removed forhistochemical examination. No accumulation of this HSA-peptide conjugatewas detectable in bone samples, whereas abundant staining was found intissue samples. Upon cryostat sections of these livers double stainingswere performed with anti-HSA antibodies and antibodies against Kupffercells (ED1), endothelial cells (RECA-1), myofibroblasts (anti-actinantibodies) or hepatic stellate cells (desmin and GFAP antibodies).Subsequently, the number of double positive cells (HSA+ and cellmarker+) were counted and related to the total number of HSA positivecells in the same area Results of the quantitative evaluation of thecarrier uptake in the liver are summarized in table 1.

TABLE 1 Relative accumulation of HSA modified with collagen VI-receptorrecognising peptides (pCVI-HSA) or PDGF receptor-recognising peptides(pPB-HSA) in non-parenchymal cells of the liver. The number ofHSA-positive cells was related to the number of cells double-positivefor HSA and a HSC marker (desmine), or a EC marker (HIS 52), or a KCmarker (ED2) or a PC marker (glycogen). % HSC % EC % KC PC pCVI-HSA 73 ±14 30 ± 10 16 ± 11 − pPB-HSA 72 ± 18 16 ± 6  11 ± 6  + HSC = hepaticstellate cells, EC = endothelial cells, KC = Kupffer cells, PC =parenchymal cells

EXAMPLE 2

Normal rats and rats with liver fibrosis (3 weeks after bile ductligation) received an intravenous injection of 10 mg/kg b.w. collagen VIreceptor-binding peptides conjugated with HSA. Based upon the results ofthe organ distribution studies with radiolabeled conjugates (FIG. 2),rats were sacrificed after ten minutes and samples of the liver and bone(from ribs, front paw, rear paw and the back) were removed forhistochemical examination. No accumulation of this HSA-peptide conjugatewas detectable in bone samples, whereas abundant staining was found intissue samples. Upon cryostat sections of these livers double stainingswere performed with anti-HSA antibodies and antibodies against Kupffercells (ED1), endothelial cells (RECA-1), myofibroblasts (anti-actinantibodies) or hepatic stellate cells (desmin and GFAP antibodies).Subsequently, the number of double positive cells (HSA+ and cellmarker+) were counted and related to the total number of HSA positivecells in the same area Results of the quantitative evaluation of thecarrier uptake in the liver are summarized in table 1.

EXAMPLE 3

Another cyclic oligopeptide recognizing the PDGF-receptor can bedescribed as follows:

-   -   -cysteine-arginine-lysine-lysine-proline-cysteine- (C*RKKPC*),        where the cysteines (C*) represent the cyclisizing residues.

Only some crucial amino acids for the PDGF-binding peptide are providedhere. The oligopeptide may be elongated without causing a change in thecellular distribution pattern in vivo. Cyclisation of this peptide canbe achieved by a disulfide bond between both cysteine groups, whereasthe free amine in one cysteine residue can be used to couple theoligopeptide to a core-molecule like albumin. Coupling of more than oneoligopeptide to albumin can be readily done.

EXAMPLE 4

A cyclic peptide which binds to the interleukin 1β receptor can bedescribed as follows:

-   -   -cysteine-aspartic        acid-lysine-proline-threonine-leucine-cysteine- (C*DKPTLC*)        where the cysteines (C*) represent the cyclisizing residues.

The receptor binding properties of the tripeptidelysine-proline-threonine (KPT) has been reported (Ferreira et al.,Nature 334: 698, 1988). Two or more additional amino acids, preferablythe two adjacent amino acids in the native interleukin 1β molecule onboth sites of this tripeptide, are in this example attached to thistripeptide. Subsequently, the terminal cysteine residues allow forcyclysation of this oligopeptide and coupling of this peptide to amacromolecule. In this way, the interleukin 1β binding site is exposedto its receptor similar to the PGDF and collagen VI receptor bindingpeptides. This conjugate may also serve as a carrier for therapeutic ordiagnostic agents for the treatment of inflammatory processes.

Description of the Drawings

FIG. 1: Organ distribution of human serum albumnin (HSA) conjugated with10 to 12 cyclic oligopeptides recognizing the PDGF-receptor in normalrats (figure A) and in rats with liver fibrosis induced by bile ductligation (3 weeks after the operation) [fig.B]. Figure C represents theorgan distribution of unmodified HSA. Organs were removed 10 minutesafter intravenous administration of radiolabeled (¹²⁵I) protein andanalyzed using a gamma-counter. The results are expressed as the mean±SD (n=3 per group). Note the accumulation of modified HSA in livers ofnormal and diseased rats, whereas native HSA remains in the blood.

FIG. 2: Organ distribution of human serum albumin (HSA) conjugated with10 to 12 cyclic oligopeptides recognizing the collagen type VI-receptorin normal rats [figure A] and in rats with liver fibrosis induced bybile duct ligation (3 weeks after the operation) [fig. B]. Organs wereremoved 10 minutes after intravenous administration of radiolabeled(¹²⁵I) protein and analyzed using a gamma-counter. The results areexpressed as the mean ±SD (n=3 per group). Note the accumulation ofmodified HSA in livers of normal and diseased rats.

FIG. 3: Intrahepatic distribution of HSA modified with 10-12 collagentype VI-receptor binding peptides in fibrotic rats (3 weeks after bileduct ligation). 10 minutes after intravenous administration of modifiedprotein, the albumin derivatives can be immunhistochemically detected ina non-parenchymal cell type of the liver using a polyclonal antibodyagainst albumin [fig. A]. The modified albumin co-localizes with themarker for HSC (desmin) [see arrowheads, fig. B].

FIG. 4: In vitro displacement of radiolabeled PDGF-BB from its receptorupon 3T3-fibroblasts by HSA-PDGF receptor-binding peptide conjugates(pPB-HSA, closed blocks), HSA (open blocks) or uncoupled PDGF-receptorbinding peptides (pPB, open circles). Note the strong inhibition ofbinding of native PDGF to fibroblasts induced by the modified HSA, butnot by native HSA or the oligopeptides alone.

Background Information to a Further Aspect of the Invention

An increased expression of the Insulin Growth FactorII/mannose-6-phosphate (IGFII/M6P) receptor has been reported uponhepatic stellate cells in particular after activation of this cell type.This led to the idea of coupling mannose-6-phosphate (M6P) to acore-protein and the use of such a neo-glycoprotein as a drug carrier toHSC. The degree of substitution of M6P to HSA necessary for this purposecould not be deduced from the present state of the art. We have foundthat a quite high degree of substitution is required for efficienttargeting. Introduction of only a few groups was not particularlysuccessful. The invention thus provides a novel type of drug carrier tothe hepatic stellate cells (HSC). The carrier can be used for thetargeting of all kinds of therapeutic agents, preferably anti-fibroticagents to HSC, or may be applied for the visualization of HSC fordiagnostic purposes.

As it was also reported that this receptor played a role in theactivation of latent TGF-beta and TGF-beta is known to be apro-fibrogenic growth factor which is a very important mediator duringfibrosis the compound acording to this further aspect of the inventionshould also be useful for diagnosis, prophylaxis and therapy of fibroticdiseases. Mannose-6-phosphate substituted proteins may also interferewith the activation of latent TGF-beta and this carrier may thereforehave an antifibrotic action of its own.

Detailed Description of the Further Aspect of the Invention

The invention in a further aspect is directed at a compound capable ofrecognising and binding a mannose 6 phosphate receptor said compoundcomprising a carrier molecule linked to a molecule capable ofrecognising and capable of binding mannose-6-phosphate receptor, saidmolecules recognising and capable of binding mannose-6-phosphatereceptor being present on the carrier molecule in at least an amountsufficient to occupy at least 20% of the carrier molecule linking sitesfor said molecules recognising and capable of bindingmannose-6-phosphate receptor, with the proviso the compound is notlatent tumor growth factor beta, thyroglobulin or a lysosomal protein.The latter are known proteins that are also known to comprise terminalmannose 6 phosphate groups and as such will bind to the mannose 6phosphate receptor. They are excluded as compounds according to theinvention. The substitution degree can be higher than 30% even as highas 40 or 50%. A suitable example of the molecule capable of recognisingand capable of binding mannose-6-phosphate receptor is mannose 6phosphate.

In a compound according to this aspect of the invention the carriermolecule can be selected from the group consisting of proteins, oligo orpolypeptides, immunoglobulins or parts thereof, oligonucleotides,disaccharides, polysaccharides, biodegradable synthetic polymers,liposomes, lipid particles, biocompatible polymers in the form ofmicrospheres or nanoparticles. The carrier molecule can suitably beselected from endogenous plasma proteins e.g. albumin, lactoferrin,alkaline phosphatase, superoxide dismutase, alpha2 macroglobulin andfibronectin. The immunoglobulins can be mono or polyclonal. Parts ofimmunoglobulins can comprise Fab′ fragments or single chain Ig.Humanised antibodies and bispecific antibodies are envisaged. Quitesuitably in a compound according to this aspect of the invention thecarrier molecule is the endogenous plasma protein albumin. A personskilled in the art will realise numerous types of carrier molecules canbe applied. They are to be pharmaceutically acceptable and of a sizesuch that they preferably are not lost due to the renal excretionthereof. Such compounds are suitably larger than 50000 Daltons.

Quite specifically in a preferred embodiment of a compound according tothis aspect of the invention at least 10 molecules capable ofrecognising and capable of binding mannose-6-phosphate receptor arelinked to the carrier molecule. The carrier based upon macromoleculessubstituted with mannose-6-phosphate residues with substitution of morethan 10 mannose-6phosphate residues per macromolecule has been foundexceptionally appropriate for proper targeting. The carrier molecule washuman serum albumin.

The invention also covers a pharmaceutical composition comprising acompound according to any of the aforementioned embodiments of thefurther aspect of the invention disclosed as targeting ingredient andany pharmaceutically acceptable carrier. A pharmaceutical compositionaccording to the invention comprises a compound in any of theembodiments mentioned above as pharmaceutically active ingredient incombination with any pharmaceutically acceptable additional carrier. Inan alternative embodiment the pharmaceutical composition can furthercomprise a drug biodegradably attached to the compound. It is alsopossible for the compound to further comprise a diagnostic markerattached thereto. A pharmaceutical composition according the inventionwill be in a medicinal dosage form. Such a dosage form can comprisesprayable, injectable or infusable solutions or solids or dosage formsfor pulmonary or other administration routes. Also a pharmaceuticalcomposition according to the invention can be in a topical form but willpreferably be in a systemically acceptable form. This means it can enterthe bloodstream without causing cloning or inadmissibly toxic reaction.

The invention is also directed at application of a compound according tothe invention in any of the abovementioned embodiments as activetargeting ingredient for manufacturing a pharmaceutical compositionaccording to the further aspect of the invention just mentioned fortherapy, prophylaxis or diagnosis of a disease selected from the groupof chronic diseases, for example fibrotic disease, sclerotic disease andchronic or acute inflammatory processes such as glomerulosclerosis,interstitial fibrosis, lung fibrosis, atherosclerosis, rheumatoidarthritis, Crohns disease, colitis ulcerosa, glomerulonephritis andsepsis.

The invention is also directed at application of a compound according tothe invention in any of the abovementioned embodiments as activetargeting ingredient for manufacturing a pharmaceutical compositionaccording to the further aspect of the invention just mentioned fortherapy, prophylaxis or diagnosis of any of the following pathologicalconditions; cell proliferation associated pathology e.g. tumors, adisease related to proliferation of HSC, fibroblast proliferationassociated pathology, endothelial cell proliferation associatedpathology and osteoblast proliferation associated pathology. Theinvention also covers a method of targeting proliferating cells,preferably tumor cells, HSC, fibroblasts, endothelial cells andosteoblasts said method comprising administration in a pharmaceuticallyacceptable amount and form of a compound or a pharmaceutical compositionaccording to the aspect of the invention just described to a subject ora tissue sample of a subject. In an alternative embodiment it alsocovers a method of targeting proliferating cells, preferably tumorcells, HSC, fibroblasts, endothelial cells and osteoblasts said methodcomprising administration in a pharmaceutically acceptable amount andform of a compound or a pharmaceutical composition according to thefurther aspect of the invention just described to a subject or a tissuesample of a subject. Alternatively it covers a method of therapy,diagnosis or prophylaxis of a disease related to proliferating cells,preferably tumor cells, HSC, fibroblasts, endothelial cells andosteoblasts, said method comprising administration in a pharmaceuticallyacceptable amount and form of a compound or a pharmaceutical compositionaccording to the further aspect of the invention to a subject or atissue sample of a subject. Specifically it covers a method of therapy,diagnosis or prophylaxis of a disease related to HSC, said methodcomprising administration in a pharmaceutically acceptable amount andform of a compound or a pharmaceutical composition as described for thefurther aspect of the invention to a subject or a tissue sample of asubject. A method of therapy, prophylaxis or diagnosis of a diseaseselected from the group consisting of fibrotic disease, scleroticdisease and chronic or acute inflammatory processes such asglomerulosclerosis, interstitial fibrosis, lung fibrosis,atherosclerosis, rheumatoid arthritis, Crohns disease, colitis ulcerosa,glomerulonephritis and sepsis, said method comprising administration ina pharmaceutically acceptable amount and form of a compound or apharmaceutical composition as described for the further aspect of theinvention to a subject or a tissue sample of a subject also falls withinthe scope of the invention.

This further aspect of the invention will be illustrated but not limitedin the following examples.

EXAMPLE 5

Mannose 6-phosphate was covalently coupled to human serum albumin (HSA)in two steps. First, p-nitrophenyl-α-D-mannopyranoside (Sigma, St.Louis, USA) was phosphorylated according to standard procedures. Themolecular weight (MW 381) and purity of the obtained crystalline productp-nitrophenyl-6-phospho-α-D-mannopyranoside was verified by massspectrometry. Subsequently, the nitro-group was reduced with 10%palladium on active carbon (Aldrich Chemie GmbH, Steinheim, Germany)under hydrogen atmosphere of 1 atm. The obtained productp-aminophenyl-6-phospho-α-D-mannopyranoside was coupled to HSA byactivation with thiophosgene. By variations in the molar ratio HSA:p-nitrophenyl-6-phospho-α-D-mannopyranoside, a series ofneoglycoproteins (M6P_(x)-HSA) were obtained, x=2, 4, 10, 21, or 28. TheM6P_(x)-HSA preparations were further purified and characterizedaccording to standard procedures. A tracer dose of modified HSA (¹²⁵Ilabelled) was intravenously administered to normal and fibrotic rats(three weeks after bile duct ligation): Ten minutes after injection ofthese compounds, rats were sacrificed and all organs were removed. Ascan also be seen in FIG. 5, the degree of substitution of mannose6-phosphate to HSA strongly influenced liver uptake. HSA with a lowdegree of sugar loading (x=2−10) accumulated for 2±1% to 9±0.5% infibrotic rat livers, while the rest of the dose remained in thecirculation. An increase in the molar ratio of M6P:HSA up to 28 caused agradual increase in liver accumulation (to 59±9% of the dose).

In addition, the intrahepatic distribution of modified HSA was examinedimmunohistochemically. Modified HSA was administered to rats (10 mg/kgb.w.) and 10 minutes after the injection samples from the liver, spleen,kidney, and bone were histochemically examined. We observed that themore mannose 6-phosphate was linked to HSA, the higher the uptake was inHSC. Quantitative evaluation of liver sections ten minutes afteradministration of modified HSA revealed that M6P10-HSA accumulated for19±10% in HSC. In contrast, 69±12% of the intrahepatic staining forM6P28-HSA was found in HSC, whereas 20±6% was found in Kupffer cells and17±6% in endothelial cells. No uptake was detected in hepatocytes andbile duct epithelial cells. Also no staining for modified HSA was foundin other organs.

EXAMPLE 6

M6P₂₁-bovine serum albumin (BSA) and M6P₂₈-HSA, synthesized andcharacterized according to standard procedures, were radiolabeled with¹²⁵I. The intrahepatic uptake of these neo-glycoproteins was measured inhuman liver slices. These slices (±10 mg liver tissue with a thicknessof approximately 10 cells) were obtained from patients with normal liverfunction and from cirrhotic patients. Significant intrahepaticaccumulation of radiolabeled BSA and HSA derivates was found within onehour after co-incubation with these slices, whereas unmodified BSA orHSA was not taken up by the human tissue samples (see FIG. 6).

EXAMPLE 7

Pyrrolidine-dithiocarbamate (PDTC, which is an inhibitor of thetranscription factor NF-kappaB) was attached to M6P₂₉-HSA by couplingthe carboxylic groups of PDTC to lysine groups of HSA according tostandard procedures. This compound was administered to rats withliverfibrosis induced by bile duct ligation. Rats receiving thisconjugate 1, 3 and 5 days after the bile duct ligation displayed lessproliferation of HSC in the parenchymal area at day 7 as compared torats receiving no treatment or PDTC alone after induction of fibrosis.HSC were demonstrated in cryostat sections with anti-desmine andanti-Glial Fibrillar Acidic Protein (GFAP) antibodies and standardindirect immunoperoxidase techniques.

Brief Description of the Drawings

FIG. 5. The organ distribution of radiolabeled M6P^(x)-HSA in fibroticrats (three weeks after bile duct ligation), 10 minutes afterintravenous administration of the modified HSA. x=2, 4,10, 21, and 28.Note that proteins substituted with 2, 4 or 10 M6P molecules per HSAremain in the blood, whereas proteins with high amounts of substitutionaccumulate in the liver.

FIG. 6. Binding and uptake of radiolabeled M6P₂₈-HSA in human livertissue at 4 degrees Celcius respectively 37 degrees Celsius.¹²⁵I-labeled modified albumin was incubated with slices (10 mg) obtainedfrom patients with a normal liver function. (TX=transplantation liver)or from patients with liver Cirrhosis (Cir).

Note the high accumulation of neo-glycoprotein in human liver slices ascompared to native HSA

1. A method for targeting Hepatic Stellate Cells (HSC) involved insclerotic and/or fibrotic diseases, and in which cells the PDGF-receptoris upregulated during disease, in a tissue sample of a subject, saidmethod comprising the steps of providing a tissue sample of a subjectand administering a carrier molecule to said tissue sample in aneffective amount, said carrier molecule being linked to at least onefurther molecule, said further molecule comprising a cyclic peptidecomprising the amino acid sequence SRNLIDC.
 2. A method for targetingHepatic Stellate Cells (HSC) involved in sclerotic and/or fibroticdiseases, and in which cells the PDGF-receptor is upregulated duringdisease, in a subject, said method comprising the steps of administeringa pharmaceutically acceptable amount and form of a carrier molecule tosaid subject being linked to at least one further molecule, said furthermolecule comprising a cyclic peptide comprising the amino acid sequenceSRNLIDC.
 3. A method according to claim 1 or 2, wherein the carriermolecule comprises additional drugs or chemicals linked thereto.
 4. Amethod according to claim 1 or 2, wherein the carrier molecule comprisesa diagnostic marker attached thereto.
 5. A method according to claim 1or 2, wherein the sclerotic or fibrotic disease is liver fibrosis.
 6. Acompound comprising a carrier molecule linked to at least one furthermolecule, said further molecule comprising a cyclic peptide wherein thecyclic portion of said cyclic peptide comprises the amino acid sequenceSRNLIDC.
 7. A compound according to claim 6, wherein in the furthermolecule, the cyclic portion of the cyclic peptide comprises multiplereceptor binding sequences.
 8. A compound according to claim 6, whereinin the further molecule, the cyclic portion of the cyclic peptidecomprises multiple receptor binding sequences directed at at least twodifferent types of receptors.
 9. A compound according to claim 6,wherein the further molecule comprises multiple cyclic peptides directedat the same or different types of receptors.
 10. A compound according toclaim 6, wherein the carrier molecule is selected from the group ofcarrier molecules consisting of proteins, oligo or polypeptides,immunoglobulins or parts thereof, oligonucleotides, disaccharides,polysaccharides, biodegradable synthetic polymers, liposomes, lipidparticles, biocompatible polymers in the form of microspheres ornanoparticles, endogenous plasma proteins, lactoferrin, alkalinephosphatasc, superoxide dismutase, alpha2 macroglobulin and fibronectin.11. A compound according to claim 6, wherein the carrier moleculecomprises additional drugs or chemicals linked thereto.
 12. A compoundaccording to claim 6, wherein the carrier molecule comprises adiagnostic marker attached thereto.
 13. A pharmaceutical compositioncomprising a compound according to any one of claims 6 or 7-12 astargeting ingredient and one or more pharmaceutically acceptablecarriers.
 14. A method for the in vitro diagnosis of liver fibrosis orkidney fibrosis comprising providing a tissue sample of a subject andadministering a compound according to any one of claims 6 or 7-12 tosaid tissue sample.
 15. A method of for preparation of a medicament forin vivo diagnosis and/or therapy of liver fibrosis or kidney fibrosiscomprising adding a suitable amount of a compound according to any oneof claims 6 or 7-12 to one or more pharmaceutically acceptable carriers.16. A composition according to claim 10, wherein said endogenous plasmaprotein is albumin.
 17. Method according to claim 14, wherein said liverfibrosis is cirrhosis, or wherein said kidney fibrosis isglomerulosclerosis or interstitial fibrosis.
 18. Method according toclaim 15, wherein said liver fibrosis is cirrhosis, or wherein saidkidney fibrosis is glomerulosclerosis or interstitial fibrosis.
 19. Amethod for targeting cells involved in sclerotic and/or fibroticdiseases selected from liver fibrosis and kidney fibrosis, and in whichcells the PDGF-receptor is upregulated during said disease, in a tissuesample of a subject, said method comprising providing said tissue sampleof a subject and administering to said tissue sample an effective amountof a carrier molecule, said carrier molecule linked to at least onefurther molecule, said further molecule comprising a cyclic peptidecomprising the amino acid sequence SRNLIDC, wherein said liver fibrosisis liver cirrhosis and said kidney fibrosis is glomerulosclerosis orinterstitial fibrosis.
 20. A method for targeting cells involved insclerotic and/or fibrotic diseases selected from liver fibrosis andkidney fibrosis, and in which cells the PDGF-receptor is upregulatedduring said disease, in a subject, said method comprising administeringa carrier molecule in a pharmaceutically acceptable amount and form tosaid subject, in a pharmaceutically acceptable amount and form, acarrier molecule, said carrier molecule linked to at least one furthermolecule, said further molecule comprising a cyclic peptide comprisingthe amino acid sequence SRNLIDC and wherein said liver fibrosis is livercirrhosis and wherein said kidney fibrosis is glomerulosclerosis orinterstitial fibrosis.
 21. A method according to claim 19 or 20, whereinthe carrier molecule comprises additional drugs or chemicals linkedthereto.
 22. A method according to claim 19 or 20, wherein the carriermolecule comprises a diagnostic marker attached thereto.